TPP-1 formulations and methods for treating CLN2 disease

ABSTRACT

Formulations comprising recombinant human tripeptidyl peptidase-1 (rhTPP1) for intrathecal, intracerebroventricular, or intraocular administration, and kits comprising the same, are disclosed. Methods of using rhTPP1 in the prevention and treatment of symptoms of Neuronal Ceroid Lipofuscinosis (CLN2) disease are also disclosed. The formulations and methods are effective in halting the progression of CLN2 disease and may be used to treat subjects having CLN2 or a family history of CLN2.

CROSS-REFERENCE TO RELATED APPLICATIONS

The benefit under 35 U.S.C. § 119(e) of U.S. Provisional PatentApplication No. 62/300,171, filed Feb. 26, 2016, and U.S. ProvisionalPatent Application No. 62/158,789, filed May 8, 2015, is hereby claimed,and the disclosure of each application is incorporated herein byreference in their entirety.

SEQUENCE LISTING

This application contains, as a separate part of the disclosure, asequence listing in computer-readable form (49443_SeqListing.txt; 8,137bytes; created Apr. 11, 2016), which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates to formulations comprising recombinanthuman tripeptidyl peptidase-1 and their use in treating Neuronal CeroidLipofuscinosis disease and associated physiological symptoms.

BACKGROUND OF THE INVENTION

Neuronal Ceroid Lipofuscinosis (CLN2) disease is a rare genetic diseasecharacterized by a deficiency of the lysosomal enzyme tripeptidylpeptidase-1 (TPP1) caused by mutations in the TPP1 gene. CLN2 disease isinherited as an autosomal recessive disorder, with an estimatedincidence of 0.5 per 100,000 live births. In the absence of TPP1,lysosomal storage materials normally metabolized by the enzymeaccumulate in many organs, and accumulation in the central nervoussystem leads to the neurodegenerative symptoms typical of CLN2 disease.The untreated disease progression of CLN2 disease has been wellcharacterized, and the natural history of the disease is remarkablyconsistent and predictable, as demonstrated by natural history data fromindependent patient populations in North America and Europe.

CLN2 disease has a predominantly ‘classic’ late infantile phenotype.Children with CLN2 disease typically develop normally until about 3years of age, when first symptoms emerge. Most commonly, CLN2 patientswill have a first unprovoked seizure and begin to lag with acquiringnormal language milestones. By age 3, all patients exhibit one or moresigns of the disease, including for example, seizures, dementia, motorloss, movement disorder, blindness, clumsiness, ataxia and cognitivedecline. From the onset of clinical symptoms, the course of the diseaseis rapid and aggressive, generally resulting in complete loss oflanguage, cognition, gait, fine motor, bulbar function and vision within2 to 4 years, rendering patients immobile, mute and blind. The patientremains in a vegetative state until death, which typically occursbetween 6 and 12 years of age.

Two quantitative rating scales have been developed by expert cliniciansin assessing the severity of CLN2 disease, and been employed in naturalhistory studies: (1) the Hamburg scale (Steinfeld et al., Am J MedGenet. 2002; 112(4):347-54); and (2) the Weill Cornell Medical College(WCMC) scale (Worgall et al., Neurology. 2007; 69(6):521-35). Thestructure and assessment methodology of the two scales is similar. Bothscales measure the loss of previously attained important neurologicalmilestones in CLN2 patients, with each unit lost in the disease ratingscale representing a fundamental milestone in progressive decline.

Analysis of disease course in untreated CLN2-affected children showsthat after the onset of disease, they predictably lose all language andgait in 3 years, with a loss of, on the average, 2.1 milestone events(i.e., 2.1 points lost in the disease rating scale) each year. Languagedecline usually precedes gait, such that the first year is characterizedby loss of intelligible speech and progression to ataxic gait, thesecond year is characterized by loss of ambulation and intelligiblelanguage, and the third year is characterized by loss of any locomotionor communication.

Recombinant human tripeptidyl peptidase-1 (rhTPP1) is being developed asa possible treatment for CLN2 disease. The rhTPP1 protein (SEQ ID NO: 1)is produced in cell culture as a zymogen (proenzyme), which does nothave enzymatic activity. The proenzyme is auto-activated at acidic pH(and by lysosomal proteases) upon uptake to the lysosome. The maturenative TPP1 protein is a lysosomal serine protease, and is the onlyknown mammalian member of the sedolisin (serine-carboxyl peptidase)family characterized by a highly conserved Ser-Glu-Asp (SED) catalytictriad. The catalytic triad on rhTPP1 is formed by S456, E253 and D341.The primary activity of the enzyme is as a tripeptidyl exopeptidase witha broad substrate specificity. Activity of the enzyme on its substrateleads to a sequential release of tripeptides from the N-terminus of theprotein substrate (Oyama et al., J Biochem. 2005; 138(2):127-34). Asecondary, significantly weaker endoproteolytic activity with a pHoptimum of 3 has also been reported (Lin et al., J Biol Chem. 2001;276(3):2249-55).

The only commercially available treatments for CLN2 are symptomatic andsupportive; there are currently no approved therapeutic options to slowor halt the inexorable progression of CLN2, much less reverse thedeleterious effects of the disease (Mole, S. E., and Williams, R. E.,2010, GeneReviews; Chang et al., in The Neuronal Ceroid Lipofuscinoses(Batten Disease); 2011, Oxford Univ Press). Preservation of motor,language and/or vision capabilities for these children would be aclinically meaningful benefit for both patients and parents/caregivers.Thus, there is a need for new treatments for CLN2 which reduce orprevent deterioration of physiological functions associated with thedisease.

SUMMARY OF THE INVENTION

The present disclosure is directed to formulations, kits, methods, andmedical uses comprising recombinant human tripeptidyl peptidase-1(rhTPP1) for treatment of Neuronal Ceroid Lipofuscinosis (CLN2) diseaseand the treatment or prevention of one or more physiological symptomsassociated with onset of the disease. In one aspect, the disclosureprovides a formulation comprising rhTPP1 for intracerebroventricular,intrathecal, or intraocular administration. Optionally, the rhTPP1comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or afragment thereof, for example, at a concentration of about 30 mg/mL inthe formulation. The formulation may have a pH of about 6.5. Theformulation may further comprise any of potassium chloride at aconcentration of about 0.01 mg/mL to about 1 mg/mL, magnesium chloridehexahydrate at a concentration of about 0.01 mg/mL to about 1 mg/mL,calcium chloride dihydrate at a concentration of about 0.01 mg/mL toabout 1 mg/mL, sodium phosphate dibasic heptahydrate at a concentrationof about 0.01 mg/mL to about 1 mg/mL, sodium phosphate monobasicmonohydrate at a concentration of about 0.01 mg/mL to about 1 mg/mL,sodium chloride at a concentration of about 1 mg/mL to about 20 mg/mL;or a combination of any or all of the foregoing. Optionally, theformulation is preservative-free and/or stable at about 5° C., e.g., forat least about 6 months. In one embodiment, the formulation comprisesrhTPP1 at a concentration of about 30 mg/mL, sodium phosphate dibasicheptahydrate at a concentration of about 0.11 mg/mL, sodium phosphatemonobasic monohydrate at a concentration of about 0.08 mg/mL, sodiumchloride at a concentration of about 8.77 mg/mL, potassium chloride at aconcentration of about 0.22 mg/mL, magnesium chloride hexahydrate at aconcentration of about 0.16 mg/mL, and calcium chloride dihydrate at aconcentration of about 0.21 mg/mL.

In another aspect, the disclosure provides a method of treating CLN2disease, or one or more symptoms associated with CLN2 disease,comprising administering about 10 mL of a composition comprising a doseof about 300 mg of rhTPP1 to a subject in need thereof, wherein thecomposition is administered to the subject over a period of about 4hours. The disclosure also provides a composition comprising rhTPP1 foruse in treating CLN2 disease, or one or more symptoms associated withCLN2 disease in a subject, comprising about 10 mL of a compositioncomprising a dose of about 300 mg of rhTPP1, wherein the dose is to beadministered to the subject over a period of about 4 hours. Thedisclosure also provides use of rhTPP1 in the manufacture of amedicament for treating CLN2 disease, or one or more symptoms associatedwith CLN2 disease, wherein the medicament comprises about 10 mL of acomposition comprising a dose of about 300 mg of rhTPP1, wherein thedose is to be administered to the subject over a period of about 4hours. In one aspect, the composition comprising rhTPP1 is administeredto the subject via a catheter. Administration of the composition isoptionally followed by flushing the catheter by administering a flushingsolution in an amount up to about 5 mL, preferably in an amount of about3 mL, more preferably in an amount of about 2 mL. The disclosure alsoprovides a method of preventing or delaying the onset of one or moresymptoms of CLN2 disease comprising administering about 10 mL of acomposition comprising a dose of about 300 mg of rhTPP1 to a subject inneed thereof, optionally wherein the subject has a family history ofCNL2 disease and wherein the composition is administered to the subjectover a period of about 4 hours. In one aspect, the compositioncomprising rhTPP1 is administered to the subject via a catheter.Administration of the composition is optionally followed by flushing thecatheter by administering a flushing solution in an amount up to about 5mL, preferably in an amount of about 3 mL, more preferably in an amountof about 2 mL. The composition may be administered intrathecally,intracerebroventricularly, and/or intraocularly, and is optionallyadministered every other week. Optionally, the composition isadministered intracerebroventricularly or intrathecally and isadministered without removal of cerebrospinal fluid from the subjectjust prior to administration of the composition comprising rhTPP1. Inanother embodiment, the composition may be administer in anisovolumetric fashion, i.e., with removal of a defined volume of CSFfrom the patient that is approximately equal to the volume of thecomposition intrathecally administered thereafter.

In one aspect, the disclosure provides a method of treating CLN2disease, or one or more symptoms associated with the disease, comprisingadministering rhTPP1 to a subject in need thereof at a dose and/orfrequency effective to maintain a physiological function or slow orreduce deterioration of a physiological function in the subject, whereinthe physiological function is language function, motor function, visionor feeding function. The disclosure also provides a compositioncomprising rhTPP1 for use in treating CLN2 disease, or one or moresymptoms associated with the disease, in a subject comprising a dose ofrhTPP1 effective to maintain a physiological function or slow or reducedeterioration of a physiological function in the subject, wherein thephysiological function is language function, motor function, vision orfeeding function. The disclosure also provides use of rhTPP1 in themanufacture of a medicament for treating CLN2 disease, or one or moresymptoms associated with the disease, wherein the medicament comprises adose of rhTPP1 effective to maintain a physiological function or slow orreduce deterioration of a physiological function in the subject, whereinthe physiological function is language function, motor function, visionor feeding function.

In another aspect, the disclosure provides a method of treating CLN2disease in a subject, or one or more symptoms associated with thedisease in a subject, comprising administering rhTPP1 to a subject inneed thereof at a dose and/or frequency effective to improvephysiological function in the subject, wherein the physiologicalfunction is language function, motor function, vision or feedingfunction. The disclosure also provides a composition comprising rhTPP1for use in treating CLN2 disease, or one or more symptoms associatedwith the disease, in a subject comprising a dose of rhTPP1 effective toimprove physiological function in the subject, wherein the physiologicalfunction is language function, motor function, vision or feedingfunction. The disclosure also provides use of rhTPP1 in the manufactureof a medicament for treating CLN2 disease, or one or more symptomsassociated with the disease, wherein the medicament comprises a dose ofrhTPP1 effective to improve physiological function in the subject,wherein the physiological function is language function, motor function,vision or feeding function. In another aspect, the disclosure provides amethod of treating CLN2 disease in a subject, or one or more symptomsassociated with the disease in a subject, comprising administeringrhTPP1 to a subject in need thereof at a dose and/or frequency effectiveto prevent or treat a neurological symptom of the disease, wherein theneurological symptom is a decrease in brain volume, a decrease in graymatter in the brain, a seizure, or an increase in cranial cerebrospinalfluid. The disclosure also provides a composition comprising rhTPP1 foruse in treating CLN2 disease, or one or more symptoms associated withthe disease, in a subject comprising a dose of rhTPP1 effective toprevent or treat a neurological symptom of the disease, wherein theneurological symptom is a decrease in brain volume, a decrease in graymatter in the brain, a seizure, or an increase in cranial cerebrospinalfluid. The disclosure also provides use of rhTPP1 in the manufacture ofa medicament for treating CLN2 disease, or one or more symptomsassociated with the disease, wherein the medicament comprises a dose ofrhTPP1 effective to prevent or treat a neurological symptom of thedisease, wherein the neurological symptom is a decrease in brain volume,a decrease in gray matter in the brain, a seizure, or an increase incranial cerebrospinal fluid. Optionally, the subject has a familyhistory of CLN2 disease.

In another aspect, the disclosure provides a method of preventing orreducing CLN2-associated motor/gait deterioration in a subjectcomprising administering a therapeutically effective dose of rhTPP1 tothe subject. The disclosure also provides a composition comprising atherapeutically effective dose of rhTPP1 for use in preventing orreducing CLN2-associated motor/gait deterioration in a subject. Thedisclosure also provides use of a therapeutically effective dose ofrhTPP1 for the manufacture of a medicament for preventing or reducingCLN2-associated motor/gait deterioration in a subject. In one aspect,the method comprises administering a dose of rhTPP1 effective to preventor reduce a decline in the subject's clinical disease rating compared toa previous rating determined before or during treatment, for example, asmeasured using a WCMC disease rating scale for gait or a Hamburg diseaserating scale for motor.

In another aspect, the disclosure provides a method of preventing orreducing CLN2-associated language deterioration in a subject comprisingadministering a therapeutically effective dose of rhTPP1 to the subject.The disclosure also provides a composition comprising a therapeuticallyeffective dose of rhTPP1 for use in preventing or reducingCLN2-associated language deterioration in a subject. The disclosure alsoprovides use of a therapeutically effective dose of rhTPP1 for themanufacture of a medicament for preventing or reducing CLN2-associatedlanguage deterioration in a subject. In one aspect, the method comprisesadministering a dose of rhTPP1 effective to prevent or reduce a declinein the subject's clinical disease rating compared to a previous ratingdetermined before or during treatment, for example, as measured using aWCMC or Hamburg disease rating scale for language.

In another aspect, the disclosure provides a method of preventing orreducing CLN2-associated vision deterioration in a subject comprisingadministering a therapeutically effective dose of rhTPP1 to the subject.The disclosure also provides a composition comprising a therapeuticallyeffective dose of rhTPP1 for use in preventing or reducingCLN2-associated vision deterioration in a subject. The disclosure alsoprovides use of a therapeutically effective dose of rhTPP1 for themanufacture of a medicament for preventing or reducing CLN2-associatedvision deterioration in a subject. In one aspect, the method comprisesadministering a dose of rhTPP1 effective to prevent or reduce a declinein the subject's clinical disease rating compared to a previous ratingdetermined before or during treatment, for example, as measured using aHamburg disease rating scale for visual function.

In another aspect, the disclosure provides a method of preventing orreducing CLN2-associated brain volume deterioration in a subjectcomprising administering a therapeutically effective dose of rhTPP1 tothe subject. The disclosure also provides a composition comprising atherapeutically effective dose of rhTPP1 for use in preventing orreducing CLN2-associated brain volume deterioration in a subject. Thedisclosure also provides use of a therapeutically effective dose ofrhTPP1 for the manufacture of a medicament for preventing or reducingCLN2-associated brain volume deterioration in a subject. In one aspect,the method comprises administering a dose of rhTPP1 effective to preventor reduce a decrease in brain volume and/or a decrease in gray mattervolume compared to a previous volume determined before or duringtreatment.

In one aspect, the disclosure provides a kit comprising a formulationcomprising rhTPP1 described herein and a flushing solution. The flushingsolution may comprise any of the formulations of the disclosure, withthe rhTPP1 omitted. For example, the flushing solution may comprisesodium phosphate dibasic heptahydrate at a concentration of about 0.11mg/mL, sodium phosphate monobasic monohydrate at a concentration ofabout 0.08 mg/mL, sodium chloride at a concentration of about 8.77mg/mL, potassium chloride at a concentration of about 0.22 mg/mL,magnesium chloride hexahydrate at a concentration of about 0.16 mg/mL,and calcium chloride dihydrate at a concentration of about 0.21 mg/mL.Optionally, the kit further comprises a reservoir for implantation and acatheter. Optionally, the kit may comprise one or more elements selectedfrom the group consisting of an extension line, an in-line filter, aport needle, at least one (optionally two) syringe(s), at least one(optionally two) syringe needle(s), and combinations thereof.

The foregoing summary is not intended to define every aspect of theinvention, and other features and advantages of the present disclosurewill become apparent from the following detailed description, includingthe drawings. The present disclosure is intended to be related as aunified document, and it should be understood that all combinations offeatures described herein are contemplated, even if the combination offeatures are not found together in the same sentence, paragraph, orsection of this disclosure. In addition, the disclosure includes, as anadditional aspect, all embodiments of the invention narrower in scope inany way than the variations specifically mentioned above. With respectto aspects of the disclosure described or claimed with “a” or “an,” itshould be understood that these terms mean “one or more” unless contextunambiguously requires a more restricted meaning. With respect toelements described as one or more within a set, it should be understoodthat all combinations within the set are contemplated. If aspects of thedisclosure are described as “comprising” a feature, embodiments also arecontemplated “consisting of” or “consisting essentially of” the feature.Additional features and variations of the disclosure will be apparent tothose skilled in the art from the entirety of this application, and allsuch features are intended as aspects of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of rhTPP1 zymogen, lacking theassociated signal peptide. The pro-segment of the enzyme is the first176 amino acid residues, and the mature enzyme is 368 amino acids inlength starting from position 177 (SEQ ID NO: 1).

FIG. 2 depicts the clinical progression of untreated subjects havingCLN2 disease in the natural history study and shows the 0 to 6 Hamburgmotor and language composite score as a function of patient age. Themedian, quartile and 10%/90% distributions; in addition to the mean and95% confidence interval, are shown.

FIGS. 3A to 3F depict the clinical assessments of 24 patients accruedover the treatment duration and show the 0 to 6 Hamburg motor andlanguage composite score. Open circles represent CLN2 scores obtained onor before the first 300 mg infusion of rhTPP1, while closed circlesrepresent CLN2 scores obtained after the first 300 mg infusion ofrhTPP1. Both the aggregate score (circles) and the contribution ofmotor/gait (squares) and language (triangles) to the aggregate score areshown. Analysis Day 1 is the date of the first infusion.

FIGS. 4A to 4I compare the change in CLN2 score from 9 patients treatedwith rhTPP1 to untreated natural history patients matched to the treatedsubjects by disease rating score (denoted with the prefix “HAM”) on the0 to 6 Hamburg motor and language aggregate scale. Results for thetreated patients are shown with a solid line in each panel compared toresults for matched, untreated natural history patients, which are shownwith a broken line.

FIG. 5 depicts the distribution of clinical change from baseline formatched, untreated natural history patients (circles) for the treatmentduration of the patient match compared to the study subjects (squares).

FIGS. 6A to 6I depict the change in CLN2 score from 9 patients treatedwith rhTPP1 to untreated natural history patients matched by diseaserating score on the 0 to 9 Hamburg motor/language/vision aggregatescale. Results for the treated patients are shown with a solid line ineach panel compared to results for matched, untreated natural historypatients, which are shown with a broken line.

FIG. 7 depicts the volume (top panel) and proportion (bottom panel) ofcerebrospinal fluid for all 24 patients measured over the treatmentduration. Each line represents one patient.

FIGS. 8A to 8L depict the brain volume of 24 treated patients. Thevolumes (top panel) and proportions (bottom panel) of white matter areshown as the difference between whole brain volume (dash-dot line) andCSF and gray matter (dashed line) and of gray matter as the differencebetween CSF and gray matter (dashed line) and CSF (solid line).

FIGS. 9A and 9B depict the average change in CLN2 score for patientstreated with rhTPP1 and untreated natural history patients. FIG. 9Adepicts the CLN2 score for 23 patients treated with 300 mg rhTPP1 for 48weeks (dashed line) and an untreated natural history cohort of 41subjects (solid line). FIG. 9B depicts the change in CLN2 score frombaseline for 23 patients treated with 300 mg rhTPP1 for 48 weeks.

FIGS. 10A to 10L depict the clinical assessments of 24 patients accruedover the treatment duration and show the 0 to 12 combined Hamburg (leftpanel) motor (squares), language (triangles), seizures (crosses), andvisual composite score (diamonds) and the 0 to 12 combined WCMC (rightpanel) gait (squares), language (triangles), myoclonus (crosses), andfeeding (diamonds) composite score. Open circles represent aggregateCLN2 scores obtained on or before the first 300 mg infusion of rhTPP1,while closed circles represent aggregate CLN2 scores obtained after thefirst 300 mg infusion of rhTPP1.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions may be useful in aiding the skilledpractitioner in understanding the disclosure. Unless otherwise definedherein, scientific and technical terms used in the present disclosureshall have the meanings that are commonly understood by those ofordinary skill in the art. Where a range of values is provided, it isunderstood that each intervening value, to the tenth of the unit of thelower limit unless the context clearly dictates otherwise, between theupper and lower limit of that range and any other stated or interveningvalue in that stated range is encompassed within the invention. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, subject to any specifically excludedlimit in the stated range.

The term “family history” refers to a subject having a blood relativediagnosed with CLN2 disease, e.g., a sibling, parent, grandparent,great-grandparent, etc.

The term “fragment” refers, in one aspect, to a recombinant proteincomprising a portion of the rhTPP1 proenzyme amino acid sequence setforth in SEQ ID NO:1 and FIG. 1. For example, a fragment may contain atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 95% of the amino acid sequence set forth in SEQID NO:1. In another aspect, a fragment may comprise the full-length (368amino acids long; amino acids 177-544 of SEQ ID NO:1) mature TPP1 enzymeamino acid sequence set forth in SEQ ID NO:2, a portion thereof, and/orat least the catalytic triad formed by the amino acid residues S456,E253, and D341. A fragment retains catalytic activity. For example, afragment exhibits tripeptidyl exopeptidase activity and/or exhibitscatalytic activity that results in the sequential release of tripeptidesfrom the N-terminus of a protein substrate. In certain aspects, a“fragment” of the rhTPP1 proenzyme comprises at least 500 consecutiveamino acids of SEQ ID NO:1, at least 450 consecutive amino acids of SEQID NO:1, at least 400 consecutive amino acids of SEQ ID NO:1, at least368 amino acids of SEQ ID NO:1, at least 350 amino acids of SEQ ID NO:1or at least 300 consecutive amino acids of SEQ ID NO:1. In otheraspects, a “fragment” of the rhTPP1 proenzyme comprises at least 350consecutive amino acids of SEQ ID NO:2, at least 325 consecutive aminoacids of SEQ ID NO:2, at least 300 consecutive amino acids of SEQ IDNO:2, at least 275 consecutive amino acids of SEQ ID NO:2, at least 250consecutive amino acids of SEQ ID NO:2 or at least 200 consecutive aminoacids of SEQ ID NO:2.

The term “intracerebroventricular” refers to administration of acomposition into the ventricular system of the brain, e.g., viainjection, infusion, or implantation (for example, into a ventricle ofthe brain).

The term “intraocular” refers to the administration of a composition tothe eye region, e.g., via injection, infusion, or implantation (forexample, into the eyeball) or topical/ophthalmic administration (forexample, using a cream, ointment, gel or liquid drops).

The term “intrathecal” refers to administration of a composition intothe lumbar region, e.g., via injection, infusion, or implantation (forexample, into the subarachnoid space of the spinal cord).

The term “therapeutically effective” refers to any therapeutic benefitthat arises as a result of the treatment methods of the presentinvention. For example, such an effect can be the beneficial effectsthat manifest in an appropriate target tissue or organ, where suchbeneficial physiological effect is compared to that physiologicalparameter being measured in the absence of the enzyme replacementtherapy. Such a therapeutic effect may be any reduction or eliminationof one or more clinical or subclinical manifestations of CLN2 disease.For example, a therapeutically effective treatment improves, reverses,delays, prevents, or reduces deterioration of one or more physiologicalfunction and/or neurological symptom of CLN2 as described herein.

The term “stable” or “stabilized” refers to a protein-containingformulation in which the protein component therein essentially retainsits physical, functional and/or chemical stability upon storage overtime. Stability can be measured at a selected temperature for a selectedtime period. Preferably, the formulation is stable at room temperature(about 30° C.) or at about 40° C. for at least 1 month and/or stable atabout 2° C. to about 8° C. for at least 1 year and preferably for atleast 2 years. For example, the extent of protein degradation oraggregation during storage can be used as an indicator of proteinstability. Thus, a “stable” formulation may be one wherein less thanabout 20%, more preferably less than about 10%, and most preferably lessthan about 5% of the protein component is present in a degraded oraggregated form in the formulation following storage. “Stable”formulations retain essentially the same functional or therapeuticcharacteristics of the newly prepared formulation. Various analyticaltechniques for measuring protein stability are available in the art andare reviewed, for example, in Peptide and Protein Drug Delivery,247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs.(1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).

The term “prevents” or “reduces” or grammatical equivalents thereof whenused in reference to the prevention or reduction of one or more symptomsor physiological consequences of CLN2 disease in a subject means thatthe rate of decline of that/those symptom(s) in the treated CLN2 subjectis slower than that observed in an untreated CLN2 subject. In thisregard, the untreated CLN2 may be the same subject that is subsequentlytreated with a composition of the present invention or may be theaverage rate of decline of the symptom(s) of interest as observed fromthe natural history study results disclosed herein.

In jurisdictions that forbid the patenting of methods that are practicedon the human body, the meaning of “administering” rhTPP1 or aformulation thereof to a human subject refers to medical uses for rhTPP1or a formulation thereof, for example, rhTPP1 or a formulation thereoffor use in treating CLN2 disease as described herein or use of rhTPP1for the manufacture of a medicament for treating CLN2 disease asdescribed herein. The broadest reasonable interpretation that isconsistent with laws or regulations defining patentable subject matteris intended. In jurisdictions that do not forbid the patenting ofmethods that are practiced on the human body, “administering” rhTPP1 ora formulation thereof includes both methods practiced on the human bodyand also the foregoing activities.

The present disclosure provides formulations and kits comprising rhTPP1,and methods of using the same to treat CLN2 disease. Administration ofrhTPP1 allows for cellular uptake of the protein by the cationindependent mannose 6 phosphate receptor (CI-MPR) and localization tothe lysosomes in cells throughout the central nervous system. The enzymeuptake into the lysosomes and subsequent activation promotes increasedcatabolism of storage material in affected tissues, reduces theprogressive accumulation of the lysosomal storage material, and arrestsdecline of the disease. The formulations and methods of the disclosureprovide therapeutic benefits that surpass those of currently approvedtreatments.

Formulations

The disclosure provides formulations comprising rhTPP1 forintracerebroventricular, intrathecal, and/or intraocular administration.In one aspect, the rhTPP1 comprises SEQ ID NO:1 or a fragment thereof.RhTPP1 proteins suitable for use in the formulations and methodsdescribed herein, and methods of obtaining the rhTPP1 proteins, aredescribed in U.S. Pat. Nos. 6,302,685 and 8,277,800, incorporated hereinby reference in their entirety.

In one aspect, the rhTPP1 comprises the amino acid sequence of SEQ IDNO:1 (amino acids 1-544 of the amino acid sequence shown in FIG. 1) or afragment thereof possessing catalytic activity. In another aspect, therhTPP1 comprises the amino acid sequence of SEQ ID NO:2 (amino acids177-544 of the amino acid sequence shown in FIG. 1) or a fragmentthereof possessing catalytic activity. In still another aspect, therhTPP1 has detectable enzyme activity or is processed in vivo to a formof the enzyme that has detectable enzyme activity (i.e., is“functional”) and has at least about 70% sequence identity with SEQ IDNO:1 or SEQ ID NO:2. For example, the functional rhTPP1 is at leastabout 70% identical, at least about 75% identical, at least about 80%identical, at least about 85% identical, at least about 90% identical,at least about 95% identical, or at least about 97% identical, to SEQ IDNO:1 or SEQ ID NO:2. In one aspect, the formulation is a liquidformulation that comprises rhTPP1 at a concentration of about 1 mg/mL toabout 100 mg/mL, for example, about 10 mg/mL to about 50 mg/mL, about 25mg/mL to about 40 mg/mL, or about 30 mg/mL to about 60 mg/mL. In variousaspects, the formulation comprises rhTPP1 at a concentration of fromabout 1 mg/mL to about 100 mg/mL, from about 5 mg/mL to about 80 mg/mL,from about 10 mg/mL to about 50 mg/mL, from about 20 mg/mL to about 40mg/mL, from about 25 mg/mL to about 35 mg/mL, more specifically about 1mg/mL, about 10 mg/ml, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL,about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, about 90mg/mL, or about 100 mg/mL. In one aspect, the formulation has a pH ofabout 5.5 to about 7.5 or about 6.0 to about 7.0, for example, about5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

In one aspect, a formulation comprising rhTPP1 of the disclosure furthercomprises one or more excipients that maintains the level of a keyelectrolyte in the cerebrospinal fluid (CSF) or ocular fluid. Forexample, in one aspect, in addition to the rhTPP1 or fragment thereof,the formulation further comprises potassium chloride at a concentrationof about 0.01 mg/mL to about 1 mg/mL, for example, about 0.1 mg/mL toabout 0.5 mg/mL, about 0.2 mg/mL to about 0.8 mg/mL, about 0.2 mg/mL toabout 0.4 mg/mL, about 0.15 mg/mL to about 0.25 mg/mL, or about 0.05mg/mL to about 0.3 mg/mL. In another aspect, the formulation furthercomprises magnesium chloride hexahydrate at a concentration of about0.01 mg/mL to about 1 mg/mL, for example, about 0.1 mg/mL to about 0.5mg/mL, about 0.1 mg/mL to about 0.8 mg/mL, about 0.1 mg/mL to about 0.3mg/mL, about 0.15 mg/mL to about 0.25 mg/mL, or about 0.05 mg/mL toabout 0.3 mg/mL. In another aspect, the formulation further comprisescalcium chloride dihydrate at a concentration of about 0.01 mg/mL toabout 1 mg/mL, for example, about 0.1 mg/mL to about 0.5 mg/mL, about0.2 mg/mL to about 0.8 mg/mL, about 0.15 mg/mL to about 0.25 mg/mL,about 0.1 mg/mL to about 0.3 mg/mL, or about 0.05 mg/mL to about 0.3mg/mL. In still another aspect, the formulation comprises a combinationof all or any of the foregoing.

In another aspect, the formulation comprising rhTPP1 further comprisesone or more buffering agents. For example, in various aspects, theformulation further comprises sodium phosphate dibasic heptahydrate at aconcentration of about 0.01 mg/mL to about 1 mg/mL, for example, about0.1 mg/mL to about 0.5 mg/mL, about 0.05 mg/mL to about 0.4 mg/mL, orabout 0.1 mg/mL to about 0.3 mg/mL; and/or sodium phosphate monobasicmonohydrate at a concentration of about 0.01 mg/mL to about 1 mg/mL, forexample, about 0.01 mg/mL to about 0.2 mg/mL, about 0.05 mg/mL to about0.3 mg/mL, or about 0.08 mg/mL to about 0.4 mg/mL.

In another aspect, the formulation further comprises an isotonicityagent, such as sodium chloride at a concentration of about 1 mg/mL toabout 20 mg/mL, for example, about 1 mg/mL to about 10 mg/mL, about 5mg/mL to about 15 mg/mL, or about 8 mg/mL to about 20 mg/mL. Otherbuffering agents and isotonicity agents known in the art are suitableand may be routinely employed for use in the formulations of the presentdisclosure.

In one aspect, a formulation comprising about 30 mg/mL of rhTPP1 furthercomprises sodium phosphate dibasic heptahydrate at a concentration ofabout 0.11 mg/mL, sodium phosphate monobasic monohydrate at aconcentration of about 0.08 mg/mL, sodium chloride at a concentration ofabout 8.77 mg/mL, potassium chloride at a concentration of about 0.22mg/mL, magnesium chloride hexahydrate at a concentration of about 0.16mg/mL, calcium chloride dihydrate at a concentration of about 0.21mg/mL, and a diluent, such as water for injection.

The rhTPP1 formulations of the present disclosure are stable and can bestored for extended periods of time without an unacceptable change inquality, potency, or purity. In one aspect, the formulation is stable ata temperature of about 5° C. (e.g., 2° C. to 8° C.) for at least 1month, for example, at least 1 month, at least 3 months, at least 6months, at least 12 months, at least 18 months, at least 24 months, ormore. In another aspect, the formulation is stable at a temperature ofless than or equal to about −20° C. for at least 6 months, for example,at least 6 months, at least 12 months, at least 18 months, at least 24months, at least 36 months, or more. In another aspect, the formulationis stable at a temperature of less than or equal to about −40° C. for atleast 6 months, for example, at least 6 months, at least 12 months, atleast 18 months, at least 24 months, at least 36 months, or more. Inanother aspect, the formulation is stable at a temperature of less thanor equal to about −60° C. for at least 6 months, for example, at least 6months, at least 12 months, at least 18 months, at least 24 months, atleast 36 months, or more.

In one aspect, a formulation of the disclosure is preservative-freeand/or stabilizer-free and thus does not contain any of thimerosal,phenylmercurate salts, chlorhexidene, phenol, benzoic acid, sorbic acid,parabens, alcohols, or other preservatives commonly found in parenteralor ophthalmic formulations.

In another aspect, the formulation of the present invention may compriseone or more preservatives, stabilizers or excipients. In this regard,numerous well known and routinely employed preservatives, stabilizersand excipients useful for protein-containing formulations forintrathecal or ICV delivery are known in the art. More specifically,examples of such additives to enzyme-containing formulations for use inintrathecal or ICV delivery are described in WO2013/096899, which isherein incorporated by reference.

Methods

The disclosure provides methods of treating CLN2 disease comprisingadministering a therapeutically effective amount of a formulationcomprising rhTPP1 described herein to a subject in need thereof. Thedisclosure also provides a composition comprising rhTPP1 for use intreating CLN2 disease described herein and use of rhTPP1 in themanufacture of a medicament for treating CLN2 disease described herein.In one aspect, severity and progression of CLN2 disease and thetherapeutic benefit of administration of rhTPP1 in a patient can bemeasured using a Hamburg or WCMC clinical disease rating scale. Both theHamburg and WCMC scales consist of four disease-related domains, whichare scored in ratings on subscales of 0 to 3 points, such that 3 pointsis normal, 2 points is abnormal but functional, 1 point is abnormal andmarkedly dysfunctional, and 0 points is no residual function. Two of thefour domains, gait/motor and language, are shared in common between thetwo scales, and have high intrinsic content validity. Each scale intotal captures changes that occur as a function of both diseaseprogression and disease management. Gait, language and vision scalescapture disease progression. Seizure frequency, movement disorders andfeeding are dependent on care decisions, particularly anticonvulsantmedications and feeding tube management. The clinical progression isoften assessed using the aggregate language and gait subscales, suchthat a rating of 6 points represents age-based normal and 0 points iscomplete loss of function. Table 1 depicts the WCMC and Hamburg CLN2disease scales.

TABLE 1 Welll Cornell Scale (WCMC) Hamburg Scale Gait 3 Normal Motor 3Walks normally 2 Abnormal but independent 2 Frequent falls, obviousclumsiness 1 Abnormal requiring assistance 1 No unaided walking orcrawling only 0 Nonambulatory 0 Immobile, mostly bedridden Language 3Normal Language 3 Normal 2 Abnormal 2 Recognizably abnormal 1 Barelyunderstandable 1 Hardly understandable 0 Unintelligible or no speech 0Unintelligible or no language Myoclonus 3 None of myoclonus, Visual 3Recognizes desirable object, (motor) chorea/tremor/athetosis, and grabsat it upgoing toes 2 One of myoclonus, 2 Grabbing for objectschorea/tremor/athetosis, or uncoordinated upgoing toes 1 Two ofmyoclonus, 1 Reacts to light chorea/tremor/authetosis, or upgoing toes 0Myoclonus and chorea/tremor/ 0 No reaction to visual stimuli athetosisand upgoing toes Feeding 3 No swallowing dysfunction Seizures 3 Noseizure in 3 months 2 Mild swallowing dysfunction 2 1-2 seizures in 3months 1 Moderate swallowing dysfunction 1 1 seizure per month 0Gastrostomy tube-dependent 0 >1 seizure per month

In various aspects, the disclosure provide a method of treating CLN2disease, or one or more clinical symptoms of CLN2 disease, comprisingadministering a composition comprising a therapeutically effectiveamount of rhTPP1 to a subject in need thereof, use of rhTPP1 in themanufacture of a medicament for the treatment of CLN2 disease in asubject, or rhTPP1 for use in treating CLN2 disease in a subject.

The disclosure also provides methods of preventing one or more clinicalsymptoms of CLN2 disease comprising administering a formulationcomprising rhTPP1 described herein to a subject in need thereof,optionally wherein the subject has a family history of CLN2 disease. Invarious aspects, the disclosure provide a method of preventing one ormore clinical symptoms of CLN2 disease comprising administering acomposition comprising a therapeutically effective amount of rhTPP1 to asubject in need thereof, use of rhTPP1 in the manufacture of amedicament for the prevention of one or more clinical symptoms of CLN2disease in a subject, or rhTPP1 for use in preventing one or moreclinical symptoms of CLN2 disease in a subject, optionally wherein thesubject has a family history of CLN2 disease.

The disclosure further provides methods of treating CLN2 diseasecomprising administering rhTPP1 to a subject in need thereof at a doseeffective to maintain a physiological function or slow or reducedeterioration of a physiological function in the subject, wherein thephysiological function is language function, motor function, vision, orfeeding function. The disclosure also provides use of rhTPP1 in themanufacture of a medicament for maintaining a physiological function orslowing or reducing deterioration of a physiological function in asubject having CLN2, and rhTPP1 for use in maintaining a physiologicalfunction or slowing or reducing deterioration of a physiologicalfunction in a subject having CLN2 disease; wherein the physiologicalfunction is language function, motor function, vision, or feedingfunction.

In one aspect, a method of treating a subject having CLN2 disease or afamily history of CLN2 disease comprises administering a dose of rhTPP1effective to maintain language function or slow or reduce deteriorationof language function to the subject. In one aspect, the deterioration oflanguage function is a reduction of at least one point compared to aprevious rating determined before or during treatment as measured usinga WCMC or Hamburg disease rating scale. In both the WCMC and Hamburgscales, a rating of 3 points indicates normal language; 2 pointsindicates (recognizably) abnormal language; 1 point indicatesbarely/hardly understandable language; and 0 points indicatesunintelligible or no language. In one aspect, the dose of rhTPP1 iseffective to maintain the subject's language rating at the same level asa previous rating determined before or during treatment, e.g., 3 points,2 points or 1 point. In another aspect, the dose of rhTPP1 is effectiveto slow or reduce CLN2-associated deterioration of language function inthe subject, which can be demonstrated by maintenance of the languagerating at the same level for a longer period of time or a smallerdecrease in the language function rating, compared to what would beexpected considering the natural progression of the disease.

In another aspect, a method of treating a subject with CLN2 or a familyhistory of CLN2 comprises administering a dose of rhTPP1 effective tomaintain motor function or slow or reduce deterioration of motorfunction to the subject. In one aspect, the deterioration of motorfunction is a reduction of at least one point compared to a previousrating determined before or during treatment as measured using a WCMC orHamburg disease rating scale. Either the clinical rating scale for gaitin the WCMC scale or for motor in the Hamburg scale can be used toevaluate motor function. In both the WCMC and Hamburg scales, a ratingof 3 points indicates normal walking; 2 points indicates abnormal butindependent walking, e.g., with frequent falls or obvious clumsiness; 1point indicates abnormal walking requiring assistance, e.g., no unaidedwalking or crawling only; and 0 points indicates the subject innon-ambulatory/immobile, e.g., mostly bedridden. In one aspect, the doseof rhTPP1 is effective to maintain the subject's motor function ratingat the same level as a previous rating determined before or duringtreatment, e.g., 3 points, 2 points, or 1 point. In another aspect, thedose of rhTPP1 is effective to slow or reduce CLN2-associateddeterioration of motor function in the subject, which can bedemonstrated by maintenance of the motor rating at the same level for alonger period of time or a smaller decrease in the motor functionrating, compared to what would be expected considering the naturalprogression of the disease.

In still another aspect, a method of treating a subject with CLN2 or afamily history of CLN2 comprises administering a dose of rhTPP1effective to maintain vision or slow or reduce deterioration of visionto the subject. In one aspect, the deterioration of vision is areduction of at least one point compared to a previous rating determinedbefore or during treatment as measured using a Hamburg disease ratingscale. According to the Hamburg scale, a rating of 3 points indicatesthe subject recognizes a desirable object and grabs at it; 2 pointsindicates grabbing for objects uncoordinated; 1 point indicates thesubject reacts to light, and 0 points indicates the subject has noreaction to visual stimuli. In one aspect, the dose of rhTPP1 iseffective to maintain the subject's vision rating at the same level as aprevious rating determined before or during treatment, e.g., 3 points, 2points or 1 point. In another aspect, the dose of rhTPP1 is effective toslow or reduce CLN2-associated deterioration of vision in the subject,which can be demonstrated by maintenance of the vision rating at thesame level for a longer period of time or a smaller decrease in thevision rating, compared to what would be expected considering thenatural progression of the disease.

In another aspect, a method of treating a subject with CLN2 or a familyhistory of CLN2 comprises administering a dose of rhTPP1 effective tomaintain feeding function or slow or reduce deterioration of feedingfunction to the subject. In one aspect, the deterioration of feedingfunction is a reduction of at least one point compared to a previousrating determined before or during treatment as measured using a WCMCrating scale. According to the WCMC scale, a rating of 3 pointsindicates no swallowing dysfunction; 2 points indicates mild swallowingdysfunction; 1 point indicates moderate swallowing dysfunction, and 0points indicates the subject is gastronomy-tube dependent. In oneaspect, the dose of rhTPP1 is effective to maintain the subject'sfeeding function rating at the same level as the previous ratingdetermined before or during treatment, e.g., 3 points, 2 points, or 1point. In another aspect, the dose of rhTPP1 is effective to slow orreduce CLN2-associated deterioration of feeding function in the subject,which can be demonstrated by maintenance of the feeding function at thesame level for a longer period of time or a smaller decrease in thefeeding rating, compared to what would be expected considering thenatural progression of the disease.

The disclosure further provides methods of treating CLN2 diseasecomprising administering rhTPP1 to a subject in need thereof at a doseeffective to improve a physiological function, wherein the physiologicalfunction is language function, motor function, vision, or feedingfunction. The disclosure also provides use of rhTPP1 in the manufactureof a medicament for improving a physiological function in a subjecthaving CLN2, or rhTPP1 for use in improving a physiological function ina subject having CLN2; wherein the physiological function is languagefunction, motor function, vision, or feeding function. Considering theprogressively degenerative nature of the disease, an improvement inlanguage function, motor function, vision, and/or feeding function,indicating that the subject has regained lost function, is especiallydesirable, but difficult to achieve with current treatment options.

In one aspect, a method of treating a subject with CLN2 diseasecomprises administering a dose of rhTPP1 effective to improve languagefunction to the subject. In one aspect, the improvement in languagefunction is an increase of at least one point compared to a previousrating determined before or during treatment as measured using a WCMC orHamburg disease rating scale. For example, a subject can improve from arating of 1 point or 2 points to a rating of 3 points, indicating areturn to normal language, or improve from a rating of 1 point to arating of 2 points.

In another aspect, a method of treating a subject with CLN2 diseasecomprises administering a dose of rhTPP1 effective to improve motorfunction to the subject. In one aspect, the improvement in motorfunction is an increase of at least one point compared to a previousrating determined before or during treatment as measured using a WCMC orHamburg disease rating scale. For example, a subject can improve from arating of 1 point or 2 points to a rating of 3 points, indicating areturn to normal walking, or improve from a rating of 1 point to arating of 2 points.

In one aspect, a method of treating a subject with CLN2 diseasecomprises administering a dose of rhTPP1 effective to improve vision tothe subject. In one aspect, the improvement in vision is an increase ofat least one point compared to a previous rating determined before orduring treatment as measured using a Hamburg disease rating scale. Forexample, a subject can improve from a rating of 1 point or 2 points to arating of 3 points, or improve from a rating of 1 point to a rating of 2points.

In another aspect, a method of treating a subject with CLN2 diseasecomprises administering a dose of rhTPP1 effective to improve feedingfunction to the subject. In one aspect, the improvement in feedingfunction is an increase of at least one point compared to a previousrating determined before or during treatment as measured using a WCMCdisease rating scale. For example, a subject can improve from a ratingof 1 point or 2 points to a rating of 3 points, indicating a return tonormal swallowing, or improve from a rating of 1 point to a rating of 2points or 3 points.

The disclosure further provides methods of treating CLN2 diseasecomprising administering rhTPP1 to a subject in need thereof at a doseeffective to prevent or treat a neurological symptom of the disease,wherein the neurological symptom is a seizure, decrease in brain volume,decrease in gray matter in the brain, or increase of cranialcerebrospinal fluid (CSF). The disclosure also provides use of rhTPP1 inthe manufacture of a medicament for preventing or treating aneurological symptom in a subject having CLN2 or a family history ofCLN2, and rhTPP1 for use in preventing or treating a neurologicalsymptom in a subject having CLN2 or a family history of CLN2; whereinthe neurological symptom is a seizure, decrease in brain volume,decrease in gray matter in the brain, or increase of cranial CSF.

In one aspect, a method of treating a subject having CLN2 or a familyhistory of CLN2 comprises administering a dose of rhTPP1 effective tomaintain or reduce the number of seizures to a subject. In one aspect,the dose is effective to reduce the number of seizures per month thatthe subject experiences. In another aspect, the dose is effective toincrease the seizure rating by at least one point compared to a previousrating determined before or during treatment as measured using a Hamburgdisease rating scale. According to the Hamburg scale, a rating of 3points indicates no seizure in 3 months; 2 points indicates 1 to 2seizures in 3 months; 1 point indicates 1 seizure per month; and 0points more than 1 seizure per month. In one aspect, the dose of rhTPP1is effective to maintain the subject's seizure rating at the same levelas the previous rating determined before or during treatment, e.g., 3points, 2 points, or one point. In another aspect, the dose of rhTPP1 iseffective to maintain or reduce the number of seizures in the subject,which can be demonstrated by maintenance of the number of seizures permonth for a longer period of time or a smaller decrease in the seizurerating, compared to what would be expected considering the naturalprogression of the disease.

In another aspect, a method of treating a subject having CLN2 or afamily history of CLN2 comprises administering a dose of rhTPP1effective to maintain brain volume or slow or reduce the decrease inbrain volume to a subject. Brain atrophy increases as the diseaseprogresses, resulting in a loss of brain volume and an associatedincrease in the volume and relative proportion of intracranial CSF.Brain volume can be measured using methods known in the art, includingimaging techniques such as magnetic resonance imaging (MRI), computedtomography (CT/CAT), positron emission tomography (PET), single photonemission computerized tomography (SPECT), electroencephalography (EEG),magnetoencephalography (MEG), and near infrared spectroscopy (NIRS). Inone aspect, the dose of rhTPP1 is effective to slow or reduce theCLN2-associated decrease in brain volume in the subject, which can bedemonstrated by maintenance of brain volume for a longer period of timeor a smaller decrease in brain volume, compared to what would beexpected considering the natural progression of the disease.

In another aspect, method of treating a subject having CLN2 or a familyhistory of CLN2 comprises administering a dose of rhTPP1 effective tomaintain gray matter in the brain or slow or reduce the decrease of graymatter in the brain to a subject. A loss of gray matter due to brainatrophy occurs as the disease progresses, resulting in a decrease ingray matter as a percentage of brain volume. The amount of gray matterin the brain can be assessed using methods known in the art, forexample, imaging techniques such as MRI, CT/CAT, PET, SPECT, EEG, MEG,and NIRS. In one aspect, the dose of rhTPP1 is effective to slow orreduce the decrease in gray matter in the subject, which can bedemonstrated by maintenance of gray matter volume for a longer period oftime or a smaller decrease in gray matter as a percentage of brainvolume, compared to what would be expected considering the naturalprogression of the disease.

In another aspect, a method of treating a subject having CLN2 or afamily history of CLN2 comprises administering a dose of rhTPP1effective to maintain the volume of cranial CSF or slow the increase inthe volume of cranial CSF to a subject. Cranial CSF increases in volumeand proportion of total CSF as a result of cerebral atrophy. The amountand proportion of cranial CSF can be assessed using methods known in theart, for example, imaging techniques such as MRI and CT/CAT. In oneaspect, the dose of rhTPP1 is effective to slow or reduce the increasein cranial CSF in the subject, which can be demonstrated by maintenanceof the volume of cranial CSF for a longer period of time or a smallerincrease in cranial CSF as a percentage of total CSF, compared to whatwould be expected considering the natural progression of the disease.

The foregoing methods, compositions for use, and uses may furthercomprise any of the following features, alone and in combination.

In one aspect, a method, composition for use, or use of the disclosurecomprises administering a formulation, composition or dose comprisingrhTPP1 to a subject continuously or continually over a period of atleast about 1 hour, for example, at least about 1 hour, at least about 2hours, at least about 3 hours, at least about 4 hours, at least about 5hours, at least about 6 hours, or more. In another aspect, a method oruse of the disclosure comprises administering a formulation, compositionor dose comprising about 20 mg to about 500 mg, about 30 mg to about 500mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 200mg to about 400 mg, about 250 mg to about 350 mg, or about 275 mg toabout 325 mg of rhTPP1 to a subject in need thereof, for example, about20 mg, about 30 mg, about 50 mg, about 100 mg, about 200 mg, about 300mg, about 400 mg, or about 500 mg of rhTPP1. In one aspect, a method oruse of the disclosure comprises administering a formulation, compositionor dose having a volume of about 20 mL or less, about 15 mL or less,about 10 mL or less, about 7.5 mL or less, or about 5 mL or less, forexample, about 20 mL, about 15 mL, about 10 mL, about 9 mL, about 8 mL,about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL about 2 mL,about 1 mL, or about 0.5 mL per dose or administration event.

In various aspects, a method, composition for use, or use of thedisclosure comprises administering a formulation, composition or dosecomprising rhTPP1 to a subject at a rate of less than or equal to about2.5 mL of the formulation, composition or dose per hour; less than orequal to about 75 mg of rhTPP1 per hour; or less than or equal to about75 mg of rhTPP1 per 2.5 mL of formulation or composition per hour. Theformulation, composition or dose is optionally administered continuouslyor continually over a period of at least about 4 hours.

In one aspect, a method, composition for use, or use of the disclosurecomprises administering a formulation, composition or dose comprisingrhTPP1 weekly or less frequently, for example, weekly, every other week,or monthly. More specifically, a method, composition for use, or use ofthe disclosure comprises administering a formulation, composition ordose comprising rhTPP1 once every 7 days, 8 days, 9 days, 10 days, 11days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27days, 28 days, 29 days, 30 days, or 31 days. In one aspect, theformulation, composition or dose is administeredintracerebroventricularly. In another aspect, the formulation,composition or dose is administered intrathecally. In still anotheraspect, the formulation, composition or dose is administeredintraocularly. In one aspect, the formulation, composition or dose isadministered intracerebroventricularly or intrathecally, as well asintraocularly. Intracerebroventricular delivery allows penetration tothe deep gray structures of the brain such as the thalami, striatum andmidbrain, due to the physiology of CSF flow in which ventriculardelivery allows flow into third and fourth ventricles, but alsopercolates through the neuropil of the cerebral hemispheres, along aslight pressure gradient from ventricle to subarachnoid space.Intrathecal and intracerebroventricular administration of recombinantenzyme to treat lysosomal storage disorders are described in U.S. Pat.No. 7,442,372, incorporated herein by reference in its entirety.

A formulation, composition, or dose of rhTPP1 of the disclosure may beadministered in a single bolus injection or series of injections (e.g.,into the brain, lumbar region, or eye), or as a continuous or continualinfusion, e.g., using an infusion pump or other implanted device. In oneaspect, a formulation, composition, or dose of rhTPP1 is administeredusing an infusion system comprising tubing, an in-line filter (e.g.,about 0.2 μm), a reservoir (e.g., intrathecal orintracerebroventricular), and a catheter. Frequently, when a compositionis administered intrathecally or intracerebroventricularly, in order toprevent adverse side effects resulting from artificially increasingintracerebral or intrathecal pressure, a volume of CSF comparable to thevolume of composition to be administered is first removed from thesubject before the composition is administered. As described in Example3, however, it is herein demonstrated that a formulation, composition,or dose of rhTPP1 of the disclosure may be administered without removalof any volume of CSF from the subject just prior to administration ofthe formulation, composition, or dose of rhTPP1.

In one aspect, a method or use of the disclosure comprises administeringabout 10 mL of a formulation, composition or dose comprising about 300mg of rhTPP1 intracerebroventricularly over a period of about 4 hoursevery other week to a subject having CLN2.

The formulations and compositions of the present invention may bedirectly administered to a subject in need (i.e., non-isovolumetric) ormay be administered subsequent to removal of a defined volume of CSFfrom the subject prior, wherein that defined volume is approximately thesame as the volume of the composition subsequently administered (i.e.,isovolumetric).

In one aspect, a method, composition for use, or use of the disclosurefurther comprises administering a flushing solution to the subjectfollowing administration of the rhTPP1. The flushing solution isadministered via the same route as the rhTPP1 and using the samedelivery system (e.g., an infusion system), to remove any rhTPP1remaining in the delivery system and to ensure the subject received thefull intended dose of rhTPP1. In one aspect, the flushing solution isadministered (e.g., using the same catheter previously used toadminister a composition comprising rhTPP1) to the subject in an amountbetween about 0.5 mL and about 5 mL, for example, about 0.5 mL, about 1mL, about 2 mL, about 3 mL, or about 5 mL. In one aspect, the flushingsolution comprises the same components as the formulation or compositioncomprising rhTPP1, but without the rhTPP1. In one aspect, the flushingsolution comprises sodium phosphate dibasic heptahydrate at aconcentration of about 0.11 mg/mL, sodium phosphate monobasicmonohydrate at a concentration of about 0.08 mg/mL, sodium chloride at aconcentration of about 8.77 mg/mL, potassium chloride at a concentrationof about 0.22 mg/mL, magnesium chloride hexahydrate at a concentrationof about 0.16 mg/mL, calcium chloride dihydrate at a concentration ofabout 0.21 mg/mL, and a diluent, such as water for injection.

Kits

The disclosure further provides kits comprising a formulation of rhTPP1described herein, in a dose and form suitable for administration to apatient. In one aspect, the kit comprises a formulation comprising about30 mg/mL of rhTPP1, sodium phosphate dibasic heptahydrate at aconcentration of about 0.11 mg/mL, sodium phosphate monobasicmonohydrate at a concentration of about 0.08 mg/mL, sodium chloride at aconcentration of about 8.77 mg/mL, potassium chloride at a concentrationof about 0.22 mg/mL, magnesium chloride hexahydrate at a concentrationof about 0.16 mg/mL, calcium chloride dihydrate at a concentration ofabout 0.21 mg/mL, and a diluent, such as water for injection. In oneaspect, the kit further comprise instructions for theintracerebroventricular, intrathecal, and/or intraocular administrationof the therapeutic compositions of the present invention, in addition tothe therapeutic formulation. In another aspect, the kit furthercomprises a flushing solution as described herein. In still anotheraspect, the kit further comprises a system for administering theformulation, comprising any or all of the following components: tubing,an in-line filter, a reservoir for implantation, and a catheter. In oneaspect, the kit may comprise catheters, reservoirs, or other devicespreloaded with the therapeutic formulations of the present disclosure.For example, catheters preloaded with about 100 mg of rhTPP1, about 200mg of rhTPP1, about 300 mg of rhTPP1, about 400 mg of rhTPP1, or about500 mg of rhTPP1, in a pharmaceutically acceptable formulation, arespecifically contemplated. Alternatively, the kit may comprisecatheters, reservoirs, or other devices that are refillable andappropriate amounts of the enzyme for refilling such devices.

In certain embodiments, kits of the present invention may comprise oneor more of the following components: an extension line (e.g., productnumber 536040, Smiths Medical, Dublin OH), an in-line filter (e.g.,product number FS116, Smiths Medical), a port needle (e.g., productnumber 21-2737-24, Smiths Medical), a syringe or two or more syringes(e.g., product number 309604, Becton Dickinson, Franklin Lakes, N.J.) ora syringe needle or two or more syringe needles (e.g., product number305196, Becton Dickinson).

The present disclosure will be more readily understood by reference tothe following Examples, which are provided by way of illustration andare not intended to be limiting.

EXAMPLES

The following Examples describe a formulation comprising rhTPP1 forintracerebroventricular (ICV) administration and the results ofadministering the formulation to human patients compared to matched,untreated natural history patients.

Example 1 Formulation of rhTPP1 for IntracerebroventricularAdministration

RhTPP1 was produced in a genetically engineered CHO host cell line andpurified by standard chromatography methods, as described in U.S. Pat.No. 6,302,685 and Sleat et al. 1997, Science 277:1802-1805, incorporatedherein by reference in their entirety. The rhTPP1 was produced as aninactive proenzyme to be auto-activated at acidic pH upon uptake to thelysosome. The proenzyme form of rhTPP1 has a calculated isotope averagemolecular weight of approximately 59 kDa. The mature enzyme has anapparent molecular weight of approximately 46 kDa. The amino acidsequence of the rhTPP1 proenzyme is set forth in SEQ ID NO:1 and shownin FIG. 1. The pro-segment of the enzyme is the first 176 amino acidresidues, and the mature enzyme is 368 amino acids in length starting atposition 177 and is set forth in SEQ ID NO:2.

The rhTPP1 formulation used in the Examples was a sterile solution forICV infusion. It was a clear and colorless to pale yellow liquidcontaining rhTPP1 protein formulated at a concentration of 30 mg/mL. Theformulation was packaged in a container closure system consisting of aType 1 clear borosilicate glass vial closed with a fluoropolymer coatedbutyl rubber stopper and capped with aluminum seal. The formulation wasstored at a temperature of −40° C.±10° C. and supplied frozen. Thetarget pH value of the formulation was pH 6.5.

The composition of the rhTPP1 formulation used in the Examples isprovided in Table 2.

TABLE 2 Concen- tration Composition Compendial Component (mg/mL) pervial Function Grade rhTPP1 30 30 mg Active NA Ingredient Sodium 0.110.11 mg Buffering Agent USP/Ph.Eur Phosphate Dibasic, HeptahydrateSodium 0.08 0.08 mg Buffering Agent USP/Ph.Eur Phosphate MonobasicMonohydrate Sodium 8.77 8.77 mg Isotonicity USP/Ph.Eur/JP Chloride AgentPotassium 0.22 0.22 mg Maintain the USP/Ph.Eur/JP Chloride level of keyCSF electrolyte Magnesium 0.16 0.16 mg Maintain the USP/Ph.Eur Chloridelevel of key Hexahydrate CSF electrolyte Calcium 0.21 0.21 mg Maintainthe USP/Ph.Eur/JP Chloride level of key Dihydrate CSF electrolyte Waterfor NA 1.00 mL Diluent USP/Ph.Eur/JP Injection (qs)

The rhTPP1 formulation was carefully designed to mimic characteristicsof human CSF, such as the concentrations of key electrolytes are similarto those found in human CSF in vivo and the formulation did not containany conventional preservatives or stabilizers as excipients. Nosignificant safety issues, i.e., serious adverse reactions, werereported or observed following administration of the rhTPP1 formulation,which could not have been previously predicted.

Stability studies were conducted at long-term (≤−60° C.) and acceleratedconditions (5±3° C.) in accordance with ICH guidelines and per protocolto monitor the time-temperature stability. Stability samples were storedin small-scale bottles composed of the same materials as the full-scalepackaging. Stability data collected on supportive and clinical batchesdemonstrated that the rhTPP1 formulation was stable at ≤−60° C. for atleast 36 months and at 5±3° C. for at least 6 months, which wassurprising considering the formulation lacked preservatives andstabilizers commonly found in pharmaceutical products. Table 3 shows theresults of the stability testing.

TABLE 3 Time Point (months) Test Specification Storage 0 3 6 9 12 18 2436 QUALITY Appearance Colorless to pale Long-term X X X X X X X X yellowliquid Accelerated X X X Sialic Acid 2-7 mol/mol Long-term X X X X X XAccelerated X X POTENCY Specific Activity, 5-15 U/mg Long-term X X X X XX X X Acid Activated Accelerated X X X Specific Activity ≤0.075 U/mgLong-term X X X X X X X X Without Acid Accelerated X X X ActivationOligosaccharide Comparable to Long-term X X X X X X X X Profilereference Accelerated X X X 10-26% bis- mannose-6- phosphateoligomannose₇ Cellular Uptake ≤10 nM Long-term X X X X X X Accelerated XX STRENGTH Protein 27-33 mg/mL Long-term X X X X X X X X ConcentrationAccelerated X X X PURITY Related Substances Report Result Long-term X XX X X X X X by RP-HPLC (% Main Peak) Accelerated X X X Molecular Size≥95% Monomer Long-term X X X X X X X X Variants Accelerated X X X ChargeComparable to Long-term X X X X X X X X Heterogeneity referenceAccelerated X X X ≥95% Main Peak Related Comparable to Long-term X X X XX X X X Substances reference Accelerated X X X COMPOSITION pH 6.0-7.0Long-term X X X X X X X X Accelerated X X X Osmolality 270-330 mOSm/KgLong-term X X X X X X X X Accelerated X X X

Example 2 Natural History Study

The quantitative assessment of CLN2 natural history disease progressionwas analyzed in a cohort of 41 untreated CLN2 patients. The Hamburgclinical scale was used for the assessment of age-appropriateneurological and functional domains affected by disease.

The quantitative description of the clinical decline in the untreatednatural history CLN2 subjects is shown in FIG. 2, The natural historyanalysis demonstrated a clear and predictable relationship of age todisease severity. After the onset of motor and language symptoms, therewas essentially a rapid linear decline in which children, on average,lost about 2 milestone events each year (linear rate of decline 2.1points per year). There was a largely predictable course, however, therewere some ‘late onset’ cases which made up less than 20% of thepopulation in the cohort. These patients tended to have later onset ofsymptoms and a longer period of mild disease, but then succumbed torapid and active decline, typically 2 to 3 years later than the classicform.

Quantitative clinical progression from the Hamburg cohort wascorroborated by superimposing the clinical ratings assessments from anindependent (patients and raters) cohort from WCMC (n=49). Althoughclinical descriptions of independent CLN2 cohorts are similar, this wasthe first confirmation of strong quantitative relationship in diseaseprogression in separate patient groups. Both cohorts of CLN2 patientshad a large majority of classic late infantile onset and progression,and a smaller proportion of children that had a ‘late onset’ phenotype,usually having early manifestations of disease at age 5 years ratherthan at 3 years. The scale using motor (gait) and language functionreproducibly captured the neurologic decline of CLN2 patients. Based onthe foregoing analyses, the cohort of natural study subjects wasdetermined to be an appropriate non-treatment control population, andthe average rate of decline of a symptom of CLN2 disease in thisuntreated, natural history population can be used as an effective andinformative comparator for any prevention or reduction in the rate ofdecline of a symptom in a subject suffering from CLN2 disease caused byadministration of a composition of the present invention.

Example 3 Phase 1/Phase 2 Open Label Dose-Escalation Study in CLN2Patients

The study was an open label treatment clinical trial to evaluate thesafety, tolerability and efficacy of a rhTPP1 formulation of thedisclosure delivered to children with CLN2 disease through an ICVcatheter at a dose of 300 mg (10 mL total volume) every other week. Thestudy was designed to assess safety and tolerability starting at lowdoses (30 mg and 100 mg), but all patients escalated to the highexpected therapeutic dose (300 mg) when the lower doses were noted by anindependent data monitoring committee to be safe. The study duration forall enrolled patients was 48 weeks of treatment at the stable expectedtherapeutic dose of 300 mg ICV every other week. The primary studyobjectives were to evaluate safety and tolerability of a rhTPP1formulation of the disclosure administered to subjects with CLN2 diseaseby an implanted ICV reservoir and cannula and to evaluate effectivenessusing a CLN2 disease-specific rating scale score in comparison withnatural history data after 12 months of treatment. The secondary studyobjectives were to evaluate the impact of treatment on measurement ofbrain atrophy in comparison with CLN2 disease natural history data after12 months of treatment.

The major inclusion criteria were a CLN2 diagnosis and an enrollment ageof at least 3 years old. Patients having a baseline disease rating scoreless than 3 at the time of screening (using the Hamburg 0 to 6 aggregatemotor/language scale) were excluded from the study. Patients less than 3year old were likely to not progress due to age rather than treatment,as depicted by the horizontal line on the progression curve. Patientswith a score at screening of 2 or less were also less linear, morevariable and considered potentially more refractory to treatment due tothe stage of disease. Thus, the treatment group was simply defined byage and score to include early and highly predictable decline.

Mean age at enrollment was 4.0 years old, slightly more girls than boys,predominantly Caucasian. The clinical CLN2 score at screening andbaseline is shown in Table 4 below, which shows the Hamburgmotor/language score for each study cohort and totals at both screeningand baseline.

TABLE 4 CLN2 Ratings - 6 point Hamburg Scale - Last Assessment withinEach Dosing Period Analysis Population: Intent-to-treat AnalysisDataset: Entire Dosing Period Stable Cohort 1 Cohort 2 Cohort 3 DosingOnly Overall (n = 3) (n = 3) (n = 3) (n = 14) (n = 23) Screening 6 1(33%) 0 1 (33%) 0 2 (9%) 5 0 0 0 2 (14%) 2 (9%) 4 0 0 0 6 (43%)  6 (26%)3 2 (67%) 3 (100%) 2 (67%) 6 (43%) 13 (57%) Study Baseline 6 1 (33%) 0 1(33%) 0 2 (9%) 5 0 0 0 2 (14%) 2 (9%) 4 0 0 0 5 (36%)  5 (22%) 3 2 (67%)3 (100%) 1 (33%) 6 (43%) 12 (52%) 2 0 0 1 (33%) 1 (7%)  2 (9%) 1 0 0 0 00 0 0 0 0 0 0

Overall, there was a skew in the pre-treatment CLN2 scores towards moreadvanced disease. Given the rapid progression of the disease and theascertainment difficulties, a skew towards the lower scores wasexpected. Further, there was some decline in the score at screening andin the period (up to two weeks) to the baseline assessment (just beforeplacement of the ICV reservoir). Four patients from the screening groupthat scored 3 slipped a point at baseline, and two patients in thescreening group that scored a 4 lost a point to 3 at baseline. The twopatients that entered as a 6 (i.e., grossly normal) were siblings ofaffected children. The disposition, demographics, and characteristics ofthe subject population are summarized in Table 5 below.

TABLE 5 Overall Disposition Subjects enrolled 24 Subjects treated 24Subjects completed 23 Subjects discontinued   1^(a) Age (years) Mean(SD) 4.3 (1.24)   Baseline CLN2 score 6  2 Sum of 5  2 Motor/Language 4 6 3 12 2  2 Mean (SD), Median 3.6 (1.06), 3.0 Genotype^(b) Common* 9(37.5%) Common × Other 8 (33.3%) Other 7 (29.2%) ^(a)Enrolled patient1287-1007 had a single dose and withdrew consent due to inability tocomply with study procedures ^(b)Similar distribution to natural historypopulation *Common Genotypes: c.622C > T and c.509-1G > C

All patients enrolled received stable dosing of 300 mg ICV every otherweek. Cohort 1 was exposed to ≥1 month at 30 mg ICV every other week,then ascended to 100 mg ICV every other week for ≥4 weeks, while Cohort2 was started on 100 mg ICV every other week for ≥4 weeks. Both Cohort 1and Cohort 2 ascended to 300 mg ICV every other week, and all subsequentpatients including Cohort 3 initiated dosing at the stable dose regimenof 300 mg ICV every other week and continued for ≥48 weeks. The 300 mgdose was administered in 10 mL infused over a period of about 4 hoursvia an ICV catheter. A volume of CSF, e.g., equivalent to the amount ofthe rhTPP1 formulation to be administered, was not removed just prior tothe start of the infusion, which was atypical, but surprisingly did notcause any adverse effects. Immediately following administration of the300 mg dose, a flushing solution in an amount of about 2 mL wasadministered to the subject via the same ICV catheter. The flushingsolution was identical to the formulation in Table 2, but did notcontain rhTPP1. The bolus dose of 300 mg of enzyme per administrationevent was significantly higher than previous intrathecal or ICVadministered enzyme replacement therapies and, as such, the safety andefficacy profiles observed after administration of such a high dose ofdrug could not have been previously predicted. More specifically,administration of a 300 mg bolus dose of rhTPP1 without associatedserious, unmanageable adverse events could not have been previouslypredicted.

Results

Effect of Treatment on Clinical Assessments of Gait and Language: Theprimary assessment tool for the quantitative evaluation of clinicalseverity was the 0 to 6 point aggregate of the gait and languagesubscales common to both the Hamburg and WCMC disease rating scales.This scale captured the predictable, rapid, and progressive clinicaldecline in matched, untreated natural history patients that was used asa comparator for the primary efficacy analysis.

The gait/language disease rating score for 23 patients with treatmentduration of more than 42 weeks is shown in FIGS. 3A to 3F. Of the 23patients, 3 patients (1244-1001, 1244-1002, and 1244-1003) were fromCohort 1 (C1), 3 patients (1244-1004, 1244-1006, and 1287-1005) werefrom Cohort 2 (C2), 3 patients (1244-1008, 1244-1009, 1244-1010) werefrom Cohort 3 (C3), and 14 patients (0119-1020, 0146-1021, 0146-1022,0146-1023, 1244-1011, 1244-1012, 1244-1017, 1244-1024, 1323-1013,1323-1014, 1323-1015, 1323-1016, 1323-1018, and 1323-1019) were from the300 mg Stable Dosing Only (SBO) group. As expected, language deficit wastypically more advanced than gait deficit. Entry scores were notrandomly distributed; 12 patients had significant disease progressionwith a combined entry score of 3 points, and 2 patients had a combinedentry score of 6 points. Given rapid progression and diseaseascertainment, children frequently present with evident decline or assiblings of those with evident decline.

Following treatment with a rhTPP1 formulation of the disclosure (shownin Table 2 above), the CLN2 gait/language disease rating score wasstabilized, as shown in FIGS. 3A to 3F. Eleven of 23 patients had nounreversed decline over the treatment period. Four patients had a singleunit decline early in the treatment period, but no un-reversed declinethereafter. Two patients (1244-1008 and 1323-1013) decreased by one unitfrom 3 to 2 points between screening and baseline, but did notexperience any additional loss in ratings while on treatment. Based onthe results, there was evident treatment benefit in all patientsregardless of cohort (starting dose) or entry score. In a number ofpatients, there were reversed ratings drops. For example, Patient1287-1005 (FIG. 3B) had a rating decrease of 2 units in the first monthof treatment, which represented a loss of function for both gait andlanguage. However, this patient regained a unit at treatment day 60, andhad no net change afterwards until analysis day 440. The regained scorewas the acquisition of language, underscoring the clinical importance ofsingle unit changes.

Neither of the 2 patients with a score of 6 at entry lost a rating unit.Seven of the 12 patients with an entry score of 3 had no unreverseddecline, and 2 were stable after an initial single unit decline. Thus,treatment benefit was evident in patients with significant deficits anddisease progression.

As demonstrated in the CLN2 natural history study, the median rate ofdecline in the untreated natural history population was estimated at 2.1units each year. Thus, all patients in the treatment group had improvedratings compared to the expected outcomes of the untreated naturalhistory population.

To establish a clearer relationship in the disease course betweentreated and matched, untreated natural history patients, each studypatient was matched with untreated natural history patients by theparameters of baseline CLN2 score, age and genotype. Although there areno clear subgroups or factors predictive of progression in CLN2 disease,these parameters are most commonly used to define disease severity.Individual treated patients were compared to each member of the naturalhistory cohort that had similar gait/language rating score at baseline,as shown in FIGS. 4A to 4I. Patients in the study were matched bybaseline CLN2 score, as follows: for a given study patient with a givenbaseline score, all natural history patients who reported one or moreCLN2 evaluations with that same CLN2 score were identified. If the studypatient's baseline CLN2 score was 2, 3, 4, or 5, then each naturalhistory patient's CLN2-vs-time profile was time-shifted left or right sothat it overlaid the study patient's baseline score. If the naturalhistory patient had multiple assessments equaling the study patient'sbaseline CLN2 score, then the mid-time point of the multiple assessmentswas used for time-shifting. If the study patient's baseline CLN2 scorewas 6 points, then the natural history patient's last score of 6 pointswas used for time-shifting. Sensitivity analyses were conducted usingother matching criteria, and results from these analyses were consistentwith the score-matched analyses.

FIGS. 4A to 4I show results from subjects treated with a rhTPP1formulation of the disclosure plotted against matched, untreated naturalhistory patients. The treated subjects and untreated natural historypatients were matched by disease rating score using the 0 to 6 unit gaitand language subscales as an aggregate. The ratings of each werecompared over the period of one year of treatment. There was a treatmentbenefit for the subjects that received rhTPP1 compared to all members ofthe matched, untreated natural history patient group. Subject 1244-1001(FIG. 4A) had a rating decrease from 3 units to 2 units after 120 daysof treatment, but regained a unit and had no net change afterwards.Subject 1244-1002 (FIG. 4B) had a rating increase from 3 units to 4units, a decrease from 4 units to 2 units, and an increase from 2 unitsto 3 units, resulting in overall maintenance of the disease rating atthe end of the study compared to Day 1. Subjects 1244-1003 (FIG. 4C) and1244-1010 (FIG. 4I) maintained a rating of 6 units, i.e., normal motorand language function, throughout the study. Subjects 1244-1004 (FIG.4D) and 1244-1009 (FIG. 4H) maintained a rating of 3 units throughoutthe study. Subject 1244-1006 (FIG. 4E) had a rating decrease from 3units to 2 units initially, but regained a unit before decreasing againfrom 3 units to 2 units with no net changes afterwards. Subject1244-1008 (FIG. 4G) had a rating decrease from 3 units to 2 unitsinitially, with no net changes afterwards.

In contrast to all of the treated subjects, the majority of theirmatched, untreated natural history patients had an unreversed ratingdecrease from 3 units to 0 units by the end of the comparative period,indicating a progression to complete lack of function for gait andlanguage combined. The matching analysis demonstrates the treatmentbenefit for those patients that maintain their disease rating score andalso that have an initial ratings drop, but subsequently stabilize.

The most complicated response (Subject 1287-1005) is shown in FIG. 4F.Although this patient dropped rapidly by 2 units from a baseline scoreof 3 points to a study score of 1 in the first month of the study, thepatient was able to regain a unit and stabilize at a score of 2 points.The interpretation of this course was clarified by comparison toscore-matched untreated natural history patients. The clinical progresswas worse in 15 of the 18 score-matched untreated natural historypatients, and the same in just 2 score-matched untreated natural historypatients (a single untreated natural history match was non-evaluable).The clinical course in untreated patients always was worsening,frequently with little time between lost milestones. There was neverre-establishment of lost function and subsequent stabilization. Matchingto the most complex treated profile was also indicative, therefore, of aclear treatment benefit.

FIG. 5 displays the distribution of clinical change from baseline inmatched, untreated natural history patients for the treatment durationof the patient match compared to the study subjects. As previouslystated, 7 of 9 (>75%) patients had no change in baseline disease ratingscale. For those 7 patients in the treatment period, all matcheduntreated natural history patients had at least a single unit decline,but more common was a multiple unit decline or 2 units to 4 units. As anexample, Patient 1244-1001 had 1 match that lost a single point, 3matches that lost 2 points and 14 matches that lost all 3 availablelanguage/gait disease rating points. Therefore, in the same period oftime, there was no change in the treated patient, but 14 of 18 (>75%)matched untreated natural history patients lost all gait and languagefunction. There was a floor effect with a baseline entry score of 3points in which many matched untreated natural history patients lost allavailable rating units, however, the 2 patients with entry scores of 6points (Patients 1244-1003 and 1244-1010) importantly showed thesematches also were actively deteriorating, some with 4 and 5 pointdeclines in the treatment period. This observation was a clear clinicaldemonstration of substantial treatment effect; most treated childrenretained entry clinical ratings in the context of untreated naturalhistory matches that actively lost language and independent gait in thesame time period, many to complete loss of function. The remaining 2treated patients that lost a single point (Subjects 1287-1005 and1287-1006) still had better clinical ratings than the vast majority ofmatches in the treatment period. In total, of the score-matcheduntreated natural history patients, 97% had worse ratings than thetreated subjects.

Using multiple matching criteria (e.g., baseline, age, and genotype),nearly 100% of comparisons showed a favorable treatment effect comparedto matched untreated natural history patients. The average treatmentdifference across matching criteria in all treated patients as comparedto natural history patients ranged from 1.9 to 2.1 points depending onthe matching criteria utilized.

FIG. 9A shows the average change in motor-language rating for patientstreated for ≥48 weeks (n=21; dashed line) and the natural history cohort(n=41; solid line). The mean decline in the disease rating for thetreated patients was 0.43 (standard deviation 0.839), with a mediandecline of 0.00 units over 48 weeks. In contrast, the mean decline inthe disease rating for the natural history cohort was 2.09 (standarddeviation 0.966), with a median decline of 1.87 units over 48 weeks.Overall, there was significant sparing (p<0.0001) of 79% of the expectedclinical decline for the treated patients. FIG. 9B shows the change inmotor-language scores for patients (n=23) from the last measurementbefore the first 300 mg dose (baseline) to the last 300 mg dose at ≥48weeks. Overall, 65% (15 out of 23) of patients either improved or had noclinical disease progression during treatment, and 87% (20 out of 23) ofthe patients performed better during treatment (i.e., had a change ofscore of −1 or greater) compared to untreated subjects from the naturalhistory study. These analyses uniformly supported the conclusion thatthere was a dramatic and clinically meaningful stabilization of CLN2score in treated patients in comparison to matched members of theuntreated natural history group, which rapidly and predictably decline.

Effect of Treatment on Clinical Assessments of Vision: In untreated CLN2patients, vision loss occurs later than the decline in language andgait, however once symptomatic, the course is predictably rapid andprogresses to blindness, Thus, preservation of vision is an importanttreatment outcome. Loss of vision can be captured on a 0 to 3 pointsubscale in a similar fashion to other subscales, in which 3 is normaland 0 is functionally blind. There was no unreversed loss in the visionsubscale domain for the majority of the treated patients during thetreatment period. When the treated patients were matched to untreatednatural history subjects by score, age and genotype using the scalecomposites of the gait, language and vision subscales (0 to 9 units), itwas clear that untreated matched natural history patients lostadditional points in comparison to the treated group.

FIGS. 6A to 6I show results from nine subjects treated with a rhTPP1formulation of the disclosure plotted against matched, untreated naturalhistory patients matched by disease rating score using the 0 to 9 unitgait, language and vision subscales as an aggregate. Subject 1244-1001(FIG. 6A) had a rating decrease of one unit from 6 to 5 points, but thenshortly regained a point to a score of 6, with no net changesafterwards. Subject 1244-1002 (FIG. 6B) had a rating increase from 5points to 6 points, followed by a decrease from 6 points to 4 points,then an increase from 4 points to 5 points, resulting in overallmaintenance of the disease rating at the end of the study compared toDay 1. Subjects 1244-1003 (FIG. 6C) and 1244-1010 (FIG. 6I) maintained arating of 9 points throughout the study, indicating normal gait,language function and vision; subjects 1244-1004 (FIG. 6D) and 1244-1009(FIG. 6H) maintained a rating of 6 points throughout the study, andsubject 1244-1008 (FIG. 6G) maintained a rating of 5 points throughoutthe study. Subject 1244-1006 (FIG. 6E) had an initial rating decrease ofone unit from 6 points to 5 points, followed by a further decrease to 4points, but regained a unit to a rating of 5 points, with no net changesthereafter. Subject 1287-1005 (FIG. 6F) had a rating decrease from 6points to 4 points, followed by an increase from 4 points to 5 pointsand a decrease to 4 points, but regained a unit again, to a final ratingof 5 points.

Addition of the vision subscale resulted in no change in the ninetreated patients over the treatment period. However, score-matched,untreated natural history patients had significant contribution ofvision loss to the aggregate score. There were multiple untreatednatural history matches with a greater than 3 point difference comparedto the treated patients, showing the contribution of vision decline tothe aggregate score over the study period. Addition of the visionsubscale thus increased the difference between treated patients andmatched untreated natural history patients. As there was no unreversedloss of a disease rating unit in the matched, untreated natural historypatients, the observation of treatment effect from rhTPP1 relating toarresting disease progression and stabilizing function can be extendedfrom motor/gait and language to include the clinical domain of vision.

Effect of Treatment on Total Disease Assessment: Patients were alsoassessed over the course of the study using a combined 12-point scalecontaining the complete Hamburg or WCMC scores. The scores on the12-point scale were the sum of the patient's individual scores for (1)motor/gait, (2) language, (3) seizures/myoclonus and (4) visual/feeding.FIGS. 10A to 10L show results from subjects treated with a rhTPP1formulation of the disclosure using the 0 to 12 unit combined Hamburg(left panel) and WCMC scale (right panel). Sixteen of 23 patients had nounreversed decline on at least one scale, and 8 had an increased scoreon at least one scale at the end of the treatment period compared tobaseline, confirming an overall treatment benefit in patients receivingrhTPP1.

Effect of Treatment on Brain Volume: MRI was used to evaluate asecondary endpoint in treated patients. The disease process ischaracterized by atrophy, cell loss and signal abnormalities. Theseparameters correlate individually or as a composite score to patient ageand disease rating score. Thus, there is a general consensus thatdisease progression correlates to MRI indices of atrophy, and multipleMRI parameters have been shown to correlate with age and diseaseseverity in CLN2 disease (Dyke et al., AJNR Am J Neuroradiol. 2013;34(4):884-9); (Paniagua et al., Clin Neuroradiol. 2013; 23(3):189-96).The imaging database that supports these conclusions is based oncross-sectional correlation of significant numbers of patients, however,there is no within-patient longitudinal acquisition of MRI images.Therefore, there is not the same ability to match longitudinalstudy-derived MRI analysis to a similarly derived natural historydatabase.

For analysis of treated patients, MRI acquisition parameters werestandardized across the hardware platforms at the study sites. Data wasacquired locally, redacted of identifying information, and transmittedto a central imaging core lab. The images were randomized such that theindependent radiologist did not know the patient or temporalrelationship to baseline. The changes in brain volume were reconstructedfrom the randomized independent central read. The data was analyzed tocompare the studies longitudinally compared to baseline across thetreated population. FIG. 7 shows the summary of MRI-measured brainvolumes in the treated patients. Cerebral atrophy results in greatervolume and proportion of intracranial CSF. An increase in thesemeasurements of atrophy is correlated to age and severity in CLN2patients. Longitudinal plots of CSF volume and proportion for thetreated patients indicated there also appeared to be little, if anychange, in measurements of CSF parameters. All patients had MRIvolumetry that appeared constant, and consistent with, the stabilizationof ratings assessments.

FIGS. 8A to 8L show the longitudinal MRI assessment of brain volumes inthe treated patients. Active neurodegeneration in CLN2 patients ischaracterized by predominant loss of gray matter and compensatory gainof CSF. However, in the assessed period, there were very stable brainvolumes and no evidence of a neurodegenerative process in treatedpatients. The volume of gray matter, shown in FIGS. 8A to 8L as thedifference between the CSF and gray matter plot (dashed line) and CSFplot (solid line) in each of the top and bottom panels, was stablethroughout the study for each of the treated patients. The change in thevolume of cortical grey matter as a percentage of total brain volumefrom the last measurement prior to the first 300 mg infusion (baseline)compared to the last observation at ≥48 weeks of treatment is shown inTable 6 below.

TABLE 6 Change from Baseline Overall to Last Observation (n = 23) N 23Mean (SD) −2.3 (2.01) Median −2.6 Min, Max −5.8, 3.1The longitudinal change of the cortical volume is −1% each year innormal children age 4-12, but −12.5% each year for untreated CLN2patients. During treatment with rhTPP1, the volume of CSF, gray matter,and white matter stayed relatively constant, attenuating 89% ofdisease-related loss of cortical volume.

Adverse Events: One patient withdrew from the study due to inability tocomply with the protocol. The remaining 23 patients remained on thestudy and tolerated treatment with the rhTPP1 drug product via the ICVroute. There were no deaths, treatment-related withdrawals, or studydiscontinuations due to a safety-related reason. Consistent with minimalimpact of device implantation, all patients were dosed within a week ofsurgery. Out of a total of 325 infusions, only 5 (1.5%) were interruptedfor any reason, with only 2 (0.6%) of these interrupted for adverseevent-related reasons. The most frequent non-CLN2 disease associatedadverse events observed in the study were pyrexia, hypersensitivity, andupper respiratory tract infection (each in 25% of subjects overall). Ingeneral, these events were mild, self-limited and managed medically.Investigator-defined hypersensitivity events were associated with fewperipheral manifestations and were medically managed with a combinationof antipyretics, antihistamine and/or steroids. Laboratory datademonstrated a lack of clinically relevant changes in peripheral labs.In the CSF, some patients had mild, transient pleocytosis with no changein CSF glucose or protein. In summary, the evaluation of safetyparameters demonstrated that treatment with a rhTPP1 formulation of thedisclosure via ICV infusion was tolerated in all patients.

Conclusion

The clinical study demonstrated that every patient with a treatmentexposure of more than 36 weeks had significant clinical benefit,characterized by complete arrest in the progression of CLN2 disease,which constituted maximal treatment benefit, as gain of function was notexpected in the time frame for patients with moderate progression andactive degeneration.

This finding was even more compelling when the patients were matched tomembers of the natural history database based on multiple parametersincluding baseline disease rating, age and genotype. This matchingrevealed that during the same period of time that treated subjectsexperienced halted disease progression on treatment with the rhTPP1 drugproduct, the matched untreated natural history patients experiencedsubstantial loss of function. All treated patients showed, therefore,arrest of disease progression compared to active disease progression inmatched untreated natural history patients. The median rate of declinein the untreated natural history population was estimated at 2.1 pointseach year based on the available natural history data, with each unit ofdecline representing a significant milestone loss of physiologicalfunction. For the majority of patients entering the study, thepreservation of 2 units translated to continued independent ambulationand meaningful communication.

Overall, the results demonstrated that a rhTPP1 formulation and methodof treatment of the disclosure has an acceptable safety/tolerabilityprofile. No subjects discontinued the study or treatment due to anadverse event. One subject withdrew from the study after one treatmentdose due to inability to comply with the protocol. Analysis of PK andimmunogenicity revealed high CNS delivery, and no formation ofantibodies in the CSF.

The foregoing Examples demonstrate that the formulations and methodscomprising rhTPP1 described herein are effective in preventing ortreating CLN2 disease and/or one or more clinical symptoms of CLN2. In adisease whose clinical course is characterized by rapid, inexorable andirreversible neurodegenerative disease progression, halting diseaseprogression, especially in each treated patient, is a substantial andunexpected clinical benefit.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this disclosure that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed:
 1. A formulation comprising non-aggregated recombinanthuman tripeptidyl peptidase-1 (rhTPP1) for intracerebroventricular,intrathecal, or intraocular administration, wherein the rhTPP1 comprisesthe amino acid sequence of SEQ ID NO: 1 or a fragment thereof, whereinthe formulation comprises rhTPP1 at a concentration of from about 25mg/mL to about 35 mg/mL, further comprising potassium chloride at aconcentration of about 0.01 mg/mL to about 1 mg/mL, magnesium chloridehexahydrate at a concentration of about 0.01 mg/mL to about 1 mg/mL, andcalcium chloride dehydrate at a concentration of about 0.01 mg/mL toabout 1 mg/mL, and wherein the formulation has a pH of 6.5 and therhTTP1 is not aggregated.
 2. The formulation of claim 1, furthercomprising sodium phosphate dibasic heptahydrate at a concentration ofabout 0.01 mg/mL to about 1 mg/mL, sodium phosphate monobasicmonohydrate at a concentration of about 0.01 mg/mL to about 1 mg/mL, andsodium chloride at a concentration of about 1 mg/mL to about 20 mg/mL.3. The formulation of claim 1, further comprising sodium phosphatedibasic heptahydrate at a concentration of about 0.11 mg/mL, sodiumphosphate monobasic monohydrate at a concentration of about 0.08 mg/mL,sodium chloride at a concentration of about 8.77 mg/mL, potassiumchloride at a concentration of about 0.22 mg/mL, magnesium chloridehexahydrate at a concentration of about 0.16 mg/mL, and calcium chloridedehydrate at a concentration of about 0.21 mg/mL.
 4. The formulation ofclaim 1, wherein the formulation is preservative-free.
 5. Theformulation of claim 1, wherein the formulation is stable at about 5° C.for at least 6 months.
 6. A kit comprising the formulation of claim 1and a flushing solution.
 7. The kit of claim 6, wherein the flushingsolution comprises sodium phosphate dibasic heptahydrate at aconcentration of about 0.11 mg/mL, sodium phosphate monobasicmonohydrate at a concentration of about 0.08 mg/mL, sodium chloride at aconcentration of about 8.77 mg/mL, potassium chloride at a concentrationof about 0.22 mg/mL, magnesium chloride hexahydrate at a concentrationof about 0.16 mg/mL, and calcium chloride dihydrate at a concentrationof about 0.21 mg/mL.
 8. The kit of claim 6, further comprising areservoir for implantation and a catheter.
 9. The kit of claim 6,further comprising one or more elements selected from the groupconsisting of an extension line, an in-line filter, a port needle, asyringe, a syringe needle, and combinations thereof.
 10. A method oftreating Neuronal Ceroid Lipofuscinosis (CLN2) disease comprisingadministering the formulation of claim 1 to a subject in need thereof.11. A method of treating Neuronal Ceroid Lipofuscinosis (CLN2) diseasecomprising administering the formula of claim 1 to a subject in needthereof at a dose of about 300 mg, wherein the dose is administered tothe subject at a rate less than or equal to 75 mg/hour.
 12. The methodof claim 11, wherein the formulation, composition or dose isadministered every other week.
 13. The method of claim 11, wherein theformulation, composition or dose is administeredintracerebroventricularly.
 14. The method of claim 11, wherein theformulation, composition or dose is administered intrathecally.
 15. Themethod of claim 11, wherein the formulation, composition or dose isadministered intraocularly.
 16. The method of claim 11, furthercomprising administering a flushing solution to the subject followingadministration of the formulation of claim
 1. 17. The method of claim16, comprising administering about 0.5 mL to about 5 mL of the flushingsolution to the subject.
 18. The method of claim 16, wherein theflushing solution comprises sodium phosphate dibasic heptahydrate at aconcentration of about 0.11 mg/mL, sodium phosphate monobasicmonohydrate at a concentration of about 0.08 mg/mL, sodium chloride at aconcentration of about 8.77 mg/mL, potassium chloride at a concentrationof about 0.22 mg/mL, magnesium chloride hexahydrate at a concentrationof about 0.16 mg/mL, and calcium chloride dihydrate at a concentrationof about 0.21 mg/mL.
 19. A method of treating Neuronal CeroidLipofuscinosis (CLN2) disease comprising administering the formulationof claim 1 to a subject in need thereof at a dose of rhTPP1 effective tomaintain a physiological function or slow or reduce deterioration of aphysiological function in the subject, wherein the physiologicalfunction is language function, motor function, vision or feedingfunction.
 20. The method of claim 19, wherein the dose of rhTTP1 iseffective to maintain language function or slow or reduce deteriorationof language function in the subject and/or maintain motor function orslow or reduce deterioration of motor function in the subject.
 21. Themethod of claim 20, wherein the deterioration of language function ormotor function is a reduction of at least one point compared to previousrating determined before or during treatment as measured using a WeillCornell Medical College or Hamburg disease rating scale.
 22. The methodof claim 19, wherein the dose of rhTPP1 is effective to maintain visionor slow or reduce deterioration of vision in the subject.
 23. The methodof claim 22, wherein the deterioration of vision is a reduction of atleast one point compared to baseline as measured using a Hamburg diseaserating scale.
 24. The method of claim 19, wherein the dose of rhTPP1 iseffective to maintain feeding function or slow or reduce deteriorationof feeding function in the subject.
 25. The method of claim 24, whereinthe deterioration of feeding function is a reduction of at least onepoint compared to baseline as measured using the Weill Cornell MedicalCollege disease rating scale.